Morphogenesis of mammalian gut endoderm

描述（由申请方提供）：肠内胚层产生许多内脏器官的主要细胞类型，包括甲状腺、胸腺、肺、胃、肝、胰腺、肠和膀胱。对正常肠道内胚层形态发生的严格理解，包括对产生肠道内胚层及其衍生组织的细胞的起源、承诺、特化和分化的了解，应该支持理解疾病进展和为这些重要器官系统设计新的治疗策略的逻辑努力。哺乳动物胚胎中胚层形成的流行观点是外胚层、中胚层和肠内胚层仅来源于原肠胚形成期间的上胚层，而胚外组织与上胚层相互作用以建立体轴，它们仅贡献于胚外结构，如卵黄囊和胎盘。肠道内胚层是产生呼吸道和消化道以及相关器官（如肺、肝和胰腺）的组织，其形态发生的细胞运动是复杂的，并且还没有得到很好的理解。使用实时成像结合遗传标记，我们已经开始研究内脏内胚层（一种假定的胚外组织）在小鼠肠道内胚层形成中的作用（图1）。我们的初步研究得出了三个关键发现。(1)一种新的小鼠肠道内胚层形态发生模型，结合了一种新的形态发生机制，导致胚胎和胚外细胞的广泛混合。(2)胚外（内脏）内胚层细胞在原肠胚期胚胎信号中心周围的典型聚集。(3)胚外组织和胚胎组织的分离是哺乳动物发育的一个基本特征，但可能并不像人们想象的那么严格，而内脏内胚层（一种专门定义为胚外组织的谱系）可能为胚胎本身提供细胞。这些问题将在以下三个具体目标中探讨。图1：结合小鼠活体成像和遗传学研究哺乳动物肠道内胚层的形成。小鼠胚胎内脏内胚层及其衍生物的活体成像和遗传标记。在胚胎第（E）6.5天，整个内脏内胚层用绿色荧光蛋白（GFP）报道分子标记。GFP蛋白揭示了在E7.5时存在于孕体的胚胎区域中的内脏内胚层来源的细胞的分散群体，其在E8.75时并入胚胎的肠管中（A，B）。具体目标1：阐明介导肠道内胚层形态发生的细胞行为。我们将使用现有的和新的报告菌株来可视化野生型胚胎和表现出内胚层缺陷的突变体中的细胞动态。具体目标二：确定导致小鼠原肠胚信号中心周围胚外（内脏）内胚层细胞组织的事件顺序，并检验这种排列对这些信号中心功能至关重要的假设。这将通过使用实时成像和分析不能产生中线结构的突变体来实现。具体目的3：确定胚胎和成年小鼠内脏内胚层和肠内胚层组织之间是否存在谱系关系。我们将确定内脏内胚层是否是胚胎和成人肠内胚层衍生组织的暂时或稳定组分。我们将使用遗传诱导的命运作图方法来检验胚外（内脏）内胚层不是唯一的胚外组织的假设。

DESCRIPTION (provided by applicant): The gut endoderm gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to understand disease progression and design new therapeutic strategies for these vital organ systems. The prevailing view of germ layer formation in mammalian embryos is that ectoderm, mesoderm and gut endoderm derive solely from the epiblast during gastrulation and while extraembryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extraembryonic structures, such as the yolk sac and placenta. The cell movements underlying the morphogenesis of the gut endoderm, the tissue that gives rise to the respiratory and digestive tracts and associated organs such as the lungs, liver and pancreas, are complex and not well understood. Using live imaging combined with genetic labeling, we have begun to investigate the role of the visceral endoderm, a presumed extraembryonic tissue, in gut endoderm formation in the mouse (Figure 1). Three key findings arise from our preliminary studies. (1) A new model for gut endoderm morphogenesis in the mouse, incorporating a novel morphogenetic mechanism resulting in extensive mixing of embryonic and extraembryonic cells. (2) The stereotypical congregation of extraembryonic (visceral) endoderm cells around signaling centers of the gastrula stage embryo. (3) The segregation of extraembryonic and embryonic tissues, a fundamental feature of mammalian development, may not be as strict as believed, and the visceral endoderm, a lineage defined as exclusively extraembryonic, may contribute cells to the embryo-proper. These questions will be explored in the following three Specific Aims. Figure 1: Combining live imaging and genetics in mice to investigate gut endoderm formation in mammals. Live imaging and genetic labeling of the visceral endoderm, and its derivatives in mouse embryos. At embryonic day (E) 6.5 the entire visceral endoderm is labeled with a green fluorescent protein (GFP) reporter. GFP protein reveals a dispersed population of visceral endoderm-derived cells present in the embryonic region of the conceptus at E7.5, which by E8.75 become incorporated into the gut tube of the embryo (A,B). Specific Aim 1: To elucidate the cell behaviors that mediate gut endoderm morphogenesis. We will use both existing and new reporter strains to visualize cell dynamics in wild type embryos and mutants that exhibit endoderm defects. Specific Aim 2: To determine the sequence of events leading to the organization of extraembryonic (visceral) endoderm cells around signaling centers of the mouse gastrula, and test the hypothesis that this arrangement is central to the function of these signaling centers. This will be achieved using live imaging and the analysis of mutants that fail to generate midline structures. Specific Aim 3: To determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. We will establish if the visceral endoderm is a transient or stable component of embryonic and adult gut endoderm derived tissues. We will test the hypothesis that extraembryonic (visceral) endoderm is not an exclusively extraembryonic tissue using a genetic induced fate mapping approach.